Of the many immunotherapy approaches under development, the ability to use bispecific antibodies or chimeric antigen receptors (CARs) to direct T-cells to selectively kill tumor cells has demonstrated significant early success. Typically, bispecific antibodies or bispecific T-cell engagers (i.e., BiTes) cross-link T-cells by binding to CD3 and to a target cancer cell surface antigen, usually through a monovalent interaction. An alternative approach is to genetically engineer a cancer patient?s T-cells to express a single chain antibody (scFv)-CD3? fusion protein that can target the cancer cell surface antigen. After re-introduction into the patient, CAR-expressing T-cells have been able to selectively eliminate the target cancer cells. While successful, the genetic engineering of cell surfaces is time consuming and irreversible and the use of BiTes requires continuous infusion. Furthermore, the resistance to both approaches due to antigen loss has been observed. Our group has shown that two dihydrofolate reductase molecules (DHFR2) fused to an ?CD3 single chain antibody (scFv) can be engineered to spontaneously self-assemble upon the addition of the chemical dimerizer, bis-methotrexate (BisMTX), into either highly stable octavalent chemically self-assembled nanorings (CSANs). CSANs have been prepared with BisMTX containing a third arm, thus enabling it to be conjugated to oligonucleotides, fluorophores, radiolabels and drugs. Recently, we have prepared ?EpCAM/?CD3 CSANs and ?CD133/?CD3-CSANs. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus forming chemically self assembled prosthetic antigen receptor (PAR) T-cells. Upon incubation of the PAR T-cells with EpCAM+ and/or CD133+ cancer cells, rapid and selective killing of primary and tumor initiating cancer stem cells (CSC) was observed. We have also demonstrated with an orthotopic murine cancer model that ?EpCAM and ?CD133 PAR T-cells are non-toxic and able in combination to eradicate tumors in vivo. A unique safety feature of our approach is the ability to remove the CSANs from the T-cells by dosing with the FDA-approved non-toxic antibiotic trimethoprim at clinically relevant concentrations, thus allowing us to deactivate the cells pharmacologically and reduce cytokine release. Consequently, as an alternative to current approaches, we determine the generality T-cell induced killing and eradication of TNBC tumors with ?EpCAM/?-CD3-CSANS and ?CD133/?CD3-CSANS. In addition, we will develop a tripspecific ?EpCAM/?CD133/?CD3 CSANs that will allow the simultaneous elimination of TNBC primary tumor cells and CSC. The successful completion of the project milestones should result in the elucidation of the key features governing a chemical biologically based non-genetic and reversible method for T-cell targeting, as well as the importance of CD133 on TNBC proliferation. These rules will be applicable to the clinical development of anti-cancer immunotherapy with greater selectivity, lower toxicity and a reduced ability for the development of resistance.